马泰勒虫新疆株EMA-1基因的原核表达及间接ELISA检测方法的建立
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摘要
马泰勒虫裂殖子表面抗原1(EMA-1)是用于马梨形虫病诊断的重要靶抗原之一。为建立实用有效的间接酶联免疫吸附试验方法,笔者根据马泰勒虫EMA-1基因序列设计并合成引物,将新疆株EMA-1全基因序列克隆于原核表达载体pGEX-4T-1上,构建pGEX-4T-1/EMA1重组质粒,转化至BL21(DE3)中,经IPTG诱导得到EMA-1融合蛋白,切胶纯化后作为包被抗原建立检测马泰勒虫抗体的rELISA方法。结果显示:(1)GST-EMA1蛋白相对分子质量56ku,与其理论值基本一致;(2)通过Western blot分析证明其具有很好的特异性和反应原性;(3)以GST-EMA1蛋白作为包被抗原建立的rELISA可明显区分马泰勒虫、驽巴贝斯虫阳性血清和健康马血清;批内和批间重复试验的最大变异系数分别为14.79%和11.06%;(4)使用建立的间接ELISA与cELISA商品试剂盒分别对新疆伊犁马场收集的96份马血清样品进行检测,检出阳性率分别为27.1%(26/96)和25.0%(24/96),两者总符合率为95.8%。结果表明,基于原核表达的GST-EMA1蛋白所建立的rELISA特异性、重复性好,检出率与马泰勒虫临床分离率接近,可为新疆马泰勒虫病(特别是隐性带虫马)的检测、监控提供有效手段。
The erythrocytic-stage surface protein,Equi Merozoite Antigen 1(EMA-1),is a major candidate for the development of a diagnostic antigen for equine piroplasmosis.The aim of the present study was to establish an indirect ELISA for practical use.According to the specific sequence of EMA-1genes of Theileria equi,apair of primers was designed and synthesized.The EMA-1gene of Xinjiang strain was cloned,and then was inserted into the prokaryotic vector pGEX-4T-1.The recombinant plasmid(pGEX-4T-1/EMA1)were transformed into Escherichia coli BL12(DE3),and the GST-EMA1 protein was obtained by induction of IPTG.The fusion protein was purified by extracting the inclusion bodies with gel slices,and the purified protein was used as coated antigen for the establishment of indirect ELISA method to detect the antibody to Theileria equi.The results indicated that the expressed EMA-1had an apparent molecular massof 56 kDa which was largely consistent with its theoretical value,and expression of protein was identified by Western blot,which confirmed that the protein had highly specificity and reactionogenicity;the purified recombinant GST-EMA1 protein was tested in an ELISA for the detection of antibodies anti-T.equi in horses,and the indirect ELISA could clearly differentiate the T.equi-infected horse sera fromBabesia caballi-infected horse sera or normal horse sera;The intraand inter-assay demonstrated that the coefficient of maximum variation was 14.79% and 11.06%respectively;96serum samples collected from horses in the state of Yili,Xinjiang were used to compare the indirect ELISA and cELISA commercial kit,the resules showed that their positive rates of T.equi infection were 27.1%(26/96)and 25.0%(24/96)respectively,and the total coincidence rate was 95.8%.These results suggest that the GST-EMA1 protein expressed in E.coli could be a reliable immunodiagnostic antigen for indirect ELISA test and that provided the means of effective detecting and monitoring for T.equi(especially in recessive infection)in Xinjiang.
The erythrocytic-stage surface protein,Equi Merozoite Antigen 1(EMA-1),is a major candidate for the development of a diagnostic antigen for equine piroplasmosis.The aim of the present study was to establish an indirect ELISA for practical use.According to the specific sequence of EMA-1genes of Theileria equi,apair of primers was designed and synthesized.The EMA-1gene of Xinjiang strain was cloned,and then was inserted into the prokaryotic vector pGEX-4T-1.The recombinant plasmid(pGEX-4T-1/EMA1)were transformed into Escherichia coli BL12(DE3),and the GST-EMA1 protein was obtained by induction of IPTG.The fusion protein was purified by extracting the inclusion bodies with gel slices,and the purified protein was used as coated antigen for the establishment of indirect ELISA method to detect the antibody to Theileria equi.The results indicated that the expressed EMA-1had an apparent molecular massof 56 kDa which was largely consistent with its theoretical value,and expression of protein was identified by Western blot,which confirmed that the protein had highly specificity and reactionogenicity;the purified recombinant GST-EMA1 protein was tested in an ELISA for the detection of antibodies anti-T.equi in horses,and the indirect ELISA could clearly differentiate the T.equi-infected horse sera fromBabesia caballi-infected horse sera or normal horse sera;The intraand inter-assay demonstrated that the coefficient of maximum variation was 14.79% and 11.06%respectively;96serum samples collected from horses in the state of Yili,Xinjiang were used to compare the indirect ELISA and cELISA commercial kit,the resules showed that their positive rates of T.equi infection were 27.1%(26/96)and 25.0%(24/96)respectively,and the total coincidence rate was 95.8%.These results suggest that the GST-EMA1 protein expressed in E.coli could be a reliable immunodiagnostic antigen for indirect ELISA test and that provided the means of effective detecting and monitoring for T.equi(especially in recessive infection)in Xinjiang.
引文
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[9]BHOORA R,BUSS P,GUTHRIE A J,et al.Genetic diversity of piroplasms in Plains zebra(Equus quagga burchellii)and Cape mountain zebra(Equus zebra zebra)in South Africa[J].Vet Parasitol,2010,174(1-2):145-149.
[10]BALDANI C D,HILARIO E,NAKAGHI A C H,et al.Production of recombinant EMA-1protein and its application for the diagnosis of Theileria equi using an enzyme immuno assay in horses from S2o Paulo State,Brazil[J].Rev Bras Parasitol Vet,2011,20(1):54-60.
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[12]GUIDI E,PRADIER S,LEBERT I,et al.Piroplasmosis in an endemic area:analysis of the risk factors and their implications in the control of Theileriosis and Babesiosis in horses[J].Parasitol Res,2015,114(1):71-83.
[13]BALDANI C D,MACHADO R Z,BOTTEON P D T L,et al.An enzyme-linked immunosorbent assay for the detection of IgG antibodies against Babesia equi in horses[J].Ciênc Rural,2004,34(5):1525-1529.
[14]JAFFER O,ABDISHAKUR F,HAKIMUDDIN F,et al.A comparative study of serological tests and PCR for the diagnosis of equine piroplasmosis[J].Parasitol Res,2010,106(3):709-713.
[15]巴音查汗.马梨形虫在新疆地区的感染现状及其防治[C]//2015中国(北京)国际马科技大会学术论文集.北京:中国畜牧兽医学会,2015:4.BA Y C H.The infection situation and control of equine piroplasmosis from Xinjiang area[C].Beijing:Chinese Association of Animal Science and Veterinary Medicine(CAAV),2015:4.(in Chinese)
[16]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.GAO S Y,ZHA E H,WANG K,et al.A‘cost-effective’method for purification of prokaryotic expression proteins in gel slices[J].Chinese Agricultural Science Bulletin,2010,26(22):24-26.(in Chinese)
[17]KUMAR S,RAKHA N K,GOYAL L,et al.Diagnostic application of recombinant equine merozoite surface antigen-1in elisa for detection of Theileria equi specific antibodies[J].Jpn J Vet Res,2015,63(3):129-137.
[18]ROSALES R,RANGEL-RIVAS A,ESCALONA A,et al.Detection of Theileria equi and Babesia caballi infections in Venezuelan horses using competitive-inhibition ELISA and PCR[J].Vet Parasitol,2013,196(1-2):37-43.
[19]VIANNA A M,GONALES R A,DE LARA A P D S S,et al.Express珘ao heteróloga da EMA-2(equi merozoite antigen)de Theileria equi em Pichia pastoris com potencial utiliza9珘ao em imunobiológicos[J].Ciênc Rural,2014,44(10):1830-1836.
[20]曹增国,王化磊,盖微微,等.埃博拉病毒抗体间接ELISA检测方法的建立及应用[J].畜牧兽医学报,2016,47(3):615-619.CAO Z G,WANG H L,GAI W W,et al.Development of an indirect ELISA for detecting Ebola virus antibody and its application[J].Acta Veterinaria et Zootechnica Sinica,2016,47(3):615-619.(in Chinese)
[21]沈婷,周斌,陈溥言.检测猪乙型脑炎病毒抗体间接ELISA方法的建立与应用[J].畜牧与兽医,2010,42(4):1-7.SHEN T,ZHOU B,CHEN P Y.Development and application of an indirect ELISA method for detection of antibody to porcine Japanese encephalitis virus[J].Animal Husbandry&Veterinary Medicine,2010,42(4):1-7.(in Chinese)
[22]宫苗苗,曾妮,程朝飞,等.狂犬病病毒基质蛋白的原核表达及其间接ELISA方法的建立[J].中国人兽共患病学报,2013,29(1):17-22.GONG M M,ZENG N,CHENG C F,et al.Development of indirect ELISA for detection of antibodies against rabies virus based on prokaryotic expression of matrix protein[J].Chinese Journal of Zoonoses,2013,29(1):17-22.(in Chinese)
[23]夏平安,尹彦涛,李素平,等.猪繁殖与呼吸综合征病毒重组N蛋白的高效表达及间接ELISA方法的建立[J].中国兽医学报,2009,29(5):537-541.XIA P A,YIN Y T,LI S P,et al.High-level expression of N protein of PRRSV Hn-1/06strain and establishment of ELISA diagnosis based on the recombinant fusion protein[J].Chinese Journal of Veterinary Science,2009,29(5):537-541.(in Chinese)
[2]KAPPMEYER L S,THIAGARAJAN M,HERNDON D R,et al.Comparative genomic analysis and phylogenetic position of Theileria equi[J].BMC Genomics,2012,13:603.
[3]DARLING S T.Equine piroplasmosis in Panama[J].J Infect Dis,1913,13(2):197-202.
[4]DARLING S T.Equine piroplasmosis in the canal zone[J].Science,1913,37(949):370-371.
[5]BRNING A,PHIPPS P,POSNETT E,et al.Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis[J].Vet Parasitol,1997,68(1-2):11-26.
[6]GARCA-BOCANEGRA I,ARENAS-MONTES A,HERNNDEZ E,et al.Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids[J].Vet J,2013,195(2):172-178.
[7]WANG M,GUO W,IGARASHI I,et al.Epidemiological investigation of equine piroplasmosis in China by enzyme-linked immunosorbent assays[J].J Vet Med Sci,2014,76(4):549-552.
[8]WISE L N,KAPPMEYER L S,MEALEY R H,et al.Review of equine piroplasmosis[J].J Vet Intern Med,2013,27(6):1334-1346.
[9]BHOORA R,BUSS P,GUTHRIE A J,et al.Genetic diversity of piroplasms in Plains zebra(Equus quagga burchellii)and Cape mountain zebra(Equus zebra zebra)in South Africa[J].Vet Parasitol,2010,174(1-2):145-149.
[10]BALDANI C D,HILARIO E,NAKAGHI A C H,et al.Production of recombinant EMA-1protein and its application for the diagnosis of Theileria equi using an enzyme immuno assay in horses from S2o Paulo State,Brazil[J].Rev Bras Parasitol Vet,2011,20(1):54-60.
[11]FRERICHS W M,HOLBROOK A A,JOHNSON A J.Equine piroplasmosis:complement-fixation titers of horses infected with Babesia caballi[J].Am J Vet Res,1969,30(5):697-702.
[12]GUIDI E,PRADIER S,LEBERT I,et al.Piroplasmosis in an endemic area:analysis of the risk factors and their implications in the control of Theileriosis and Babesiosis in horses[J].Parasitol Res,2015,114(1):71-83.
[13]BALDANI C D,MACHADO R Z,BOTTEON P D T L,et al.An enzyme-linked immunosorbent assay for the detection of IgG antibodies against Babesia equi in horses[J].Ciênc Rural,2004,34(5):1525-1529.
[14]JAFFER O,ABDISHAKUR F,HAKIMUDDIN F,et al.A comparative study of serological tests and PCR for the diagnosis of equine piroplasmosis[J].Parasitol Res,2010,106(3):709-713.
[15]巴音查汗.马梨形虫在新疆地区的感染现状及其防治[C]//2015中国(北京)国际马科技大会学术论文集.北京:中国畜牧兽医学会,2015:4.BA Y C H.The infection situation and control of equine piroplasmosis from Xinjiang area[C].Beijing:Chinese Association of Animal Science and Veterinary Medicine(CAAV),2015:4.(in Chinese)
[16]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.GAO S Y,ZHA E H,WANG K,et al.A‘cost-effective’method for purification of prokaryotic expression proteins in gel slices[J].Chinese Agricultural Science Bulletin,2010,26(22):24-26.(in Chinese)
[17]KUMAR S,RAKHA N K,GOYAL L,et al.Diagnostic application of recombinant equine merozoite surface antigen-1in elisa for detection of Theileria equi specific antibodies[J].Jpn J Vet Res,2015,63(3):129-137.
[18]ROSALES R,RANGEL-RIVAS A,ESCALONA A,et al.Detection of Theileria equi and Babesia caballi infections in Venezuelan horses using competitive-inhibition ELISA and PCR[J].Vet Parasitol,2013,196(1-2):37-43.
[19]VIANNA A M,GONALES R A,DE LARA A P D S S,et al.Express珘ao heteróloga da EMA-2(equi merozoite antigen)de Theileria equi em Pichia pastoris com potencial utiliza9珘ao em imunobiológicos[J].Ciênc Rural,2014,44(10):1830-1836.
[20]曹增国,王化磊,盖微微,等.埃博拉病毒抗体间接ELISA检测方法的建立及应用[J].畜牧兽医学报,2016,47(3):615-619.CAO Z G,WANG H L,GAI W W,et al.Development of an indirect ELISA for detecting Ebola virus antibody and its application[J].Acta Veterinaria et Zootechnica Sinica,2016,47(3):615-619.(in Chinese)
[21]沈婷,周斌,陈溥言.检测猪乙型脑炎病毒抗体间接ELISA方法的建立与应用[J].畜牧与兽医,2010,42(4):1-7.SHEN T,ZHOU B,CHEN P Y.Development and application of an indirect ELISA method for detection of antibody to porcine Japanese encephalitis virus[J].Animal Husbandry&Veterinary Medicine,2010,42(4):1-7.(in Chinese)
[22]宫苗苗,曾妮,程朝飞,等.狂犬病病毒基质蛋白的原核表达及其间接ELISA方法的建立[J].中国人兽共患病学报,2013,29(1):17-22.GONG M M,ZENG N,CHENG C F,et al.Development of indirect ELISA for detection of antibodies against rabies virus based on prokaryotic expression of matrix protein[J].Chinese Journal of Zoonoses,2013,29(1):17-22.(in Chinese)
[23]夏平安,尹彦涛,李素平,等.猪繁殖与呼吸综合征病毒重组N蛋白的高效表达及间接ELISA方法的建立[J].中国兽医学报,2009,29(5):537-541.XIA P A,YIN Y T,LI S P,et al.High-level expression of N protein of PRRSV Hn-1/06strain and establishment of ELISA diagnosis based on the recombinant fusion protein[J].Chinese Journal of Veterinary Science,2009,29(5):537-541.(in Chinese)